Methods and compositions for diagnosing and treating glaucoma

ABSTRACT

Provided herein are methods of treating glaucoma in a patient, comprising: obtaining a biological sample from the patient; testing the biological sample for presence of a mutation in Kir6.2 protein or KCNJ11 gene and a mutation in the Aquaporin-9 protein or AQP-9 gene; and provided that the biological sample tests positive for the presence of a mutation in Kir6.2 protein or KCNJ11 gene and a mutation in the AQP-9 gene or aquaporin-9 protein, administering to the patient a therapeutically effective amount of a sulfonylurea such as tolbutamide or a physiologically equivalent salt or solvate thereof and a pharmaceutically acceptable carrier. Also provided herein are methods of maintaining and/or improving eye health in a subject, comprising: administering to the patient a therapeutically effective amount of tolbutamide or a physiologically equivalent salt or solvate thereof, and a pharmaceutically acceptable carrier.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of U.S. patent applicationSer. No. 15/970,776, which claims the benefit of priority to U.S.Provisional Patent Application Ser. No. 62/502,268 filed May 5, 2017.The entire contents of each of the above applications are incorporatedherein by reference.

FIELD OF THE INVENTION

The present disclosure relates to eye care products, particularly as itrelates to glaucoma.

BACKGROUND OF THE DISCLOSURE

All publications herein are incorporated by reference to the same extentas if each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Thefollowing description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Aqueous humor is a transparent, watery fluid similar to plasma, which issecreted from the ciliary epithelium. It's made up of 99.9% water—theother 0.1% consists of sugars, vitamins, proteins and other nutrients.It fills both the anterior and the posterior chambers of the eye. Thisfluid nourishes the cornea and the lens, as well as giving the eye itsshape.

The aqueous humor plays an essential role in the health of the eye. Aswell as nourishing the cornea and the lens by supplying nutrition suchas amino acids and glucose, the aqueous humor maintains intraocularpressure, transports vitamin C in the anterior segment to act as ananti-oxidant agent, and provides inflation for expansion of the cornea,which in turn protects against dust, wind, pollen grains, and a numberof pathogens. Thus, continuous production of aqueous humor is criticalin ensuring that the optical physics and health of the eye are properlymaintained.

Currently drugs to treat glaucoma either suppress aqueous formation,e.g. beta blockers, or increase outflow of aqueous usually by thenon-canonical uveoscleral pathway, e.g. prostaglandins. Suppressingaqueous formation will reduce nutrients to the eyes whereas increasingaqueous outflow alone does not bring needed nutrients to the eye and maynot be sufficient to eliminate harmful metabolic waste as evidenced bythe slower, but continuing progression of vision loss in glaucomapatients.

Production of aqueous humor in the eye may be affected due to severalreasons, such as, for example, glaucoma, cataract, old age, etc. Thus,there remains a need in the art for new compositions improving eyehealth by regulating the dynamics of aqueous humor by increasing aqueousproduction to better supply the eye with nutrients while at the sametime increasing aqueous flow out of the eye to maintain normalintraocular pressure and to eliminate metabolic waste products that canbe harmful and lead to retinal neurodegeneration as in glaucoma.

SUMMARY OF THE DISCLOSURE

In one aspect, disclosed herein is a method of diagnosing glaucoma in apatient in need thereof, comprising: determining if the rs5215 GTC->ATCsingle nucleotide polymorphism (SNP) is present in the KCNJ11 gene,which replaces the amino acid valine with isoleucine at position 337(V337I) of Kir6.2 subunit of the ATP-sensitive potassium (KATP) channel;determining if the rs1867380 ACA->GCA SNP is present in the aquaporin-9(AQP9) gene, which replaces the amino acid threonine with alanine atposition 279 (T279A) of AQP9; and diagnosing glaucoma in the patientwhen one or both of V337I and T279A mutations are present in thepatient. In various embodiments, the glaucoma may be normal tension openangle glaucoma, high tension open angle glaucoma, and/or exfoliativeglaucoma.

In one embodiment, the method further comprises administering atreatment to the patient for the treatment of glaucoma. Preferably, thetreatment comprises an ophthalmically acceptable formulation of aninhibitor of the KATP channel. The inhibitor of the KATP channel may besulfonylurea, such as tolbutamide. In some embodiments, tolbutamide isadministered in a concentration of 1 μg-10 μg, or 10 μg-50 μg, or 50μg-100 μg, or 100 μg-200 μg, or 200 μg-300 μg, or 300 μg-400 μg, or 400μg-500 μg, or 500 μg-600 μg, or 600 μg-700 μg, or 700 μg-800 μg, or 800μg-900 μg, or 900 μg-1000 μg. In some embodiments, tolbutamide ispresent in the ophthalmically acceptable formulation at a concentrationof 0.01-0.1%, or 0.1-0.5%, or 0.5-0.9% (w/v). In other embodiments, theinhibitor of the KATP channel is a glinide, memantine, and/orchlorpromazine.

In one embodiment, the inhibitor of the KATP channel is administeredtopically to the eye. In another embodiment, the inhibitor of the KATPchannel is administered as a slow-release ocular insert. In yet anotherembodiment, the inhibitor of the KATP channel is administered byinjection into the eye

In another aspect, disclosed herein is a method of increasing aqueoushumor production in a patient in need thereof, comprising: identifying apatient having (a) nonsense mutation rs5215 in the KCNJ11 gene, whichreplaces the amino acid Valine with Isoleucine at position 337 (V337I)of the Kir 6.2 protein and/or (b) nonsense mutation rs1867380 in theAQP9 gene, which replaces amino acid threonine with alanine at position279 (T279A) of aquaporin-9; and administering to the patient upon,identification of one or both of V337I and T279A mutations, acomposition comprising tolbutamide or a physiologically equivalent saltor solvate thereof and a pharmaceutically acceptable carrier, whereinthe administration of tolbutamide increases the aqueous humorproduction.

In another aspect, disclosed herein is a method of increasingintracellular and decreasing extracellular potassium in the trabecularmeshwork cells of glaucoma patients, comprising: identifying a patienthaving (a) nonsense mutation rs5215 in the KCNJ11 gene, which replacesthe amino acid Valine with Isoleucine at position 337 (V337I)) of theKir 6.2 protein and/or (b) nonsense mutation rs1867380 in the AQP9 gene,which replaces the amino acid threonine with alanine at position 279(T279A) of aquaporin-9; administering to the eye of the patient upon,identification of the mutation, a composition comprising a sulfonylureaat a concentration of 0.1-0.9% (w/v) or a physiologically equivalentsalt or solvate thereof and a pharmaceutically acceptable carrier,wherein the administration of tolbutamide increases the aqueous humorproduction, and wherein administration of the sulfonylurea increasingintracellular and decreasing extracellular potassium in the trabecularmeshwork cells of glaucoma patients.

Various embodiments disclosed herein include a method of treatingglaucoma in a patient, comprising: obtaining a biological sample fromthe patient; testing the biological sample for presence of a mutation inKir6.2 protein or KCNJ11 gene and a mutation in the aquaporin-9 proteinor the AQP-9 gene; and provided that the biological sample testspositive for the presence of a mutation in Kir6.2 protein or KCNJ11 geneand/or a mutation in the aquaporin-9 protein or the AQP-9 gene,administering to the eye of the patient a composition comprising atherapeutically effective amount of tolbutamide or other sulfonylureacompound or a physiologically equivalent salt or solvate thereof and apharmaceutically acceptable carrier, and wherein tolbutamide or othersulfonylurea is at a concentration of 0.001-2% (w/v). In one embodiment,the mutation is a nonsense mutation, rs5215 (Gtc/Atc), in the KCNJ11gene that replaces valine with isoleucine (V337I) at position 337 of theKir6.2 protein subunit of the ATP-sensitive potassium channels. In oneembodiment, the mutation is a rs1867380 (Aca/Gca) in the AQP9 gene thatresults in the substitution of threonine for alanine (T279A) at position279 of the aquaporin-9 protein. In one embodiment, the presence of amutation is determined using a single nucleotide polymorphism (SNP)genotyping method. In one embodiment, the pharmaceutically acceptablecarrier is an ophthalmically acceptable carrier. In one embodiment, thedosages are administered from 1 to 4 times per day. In one embodiment,the method further comprises administration by topical application tothe eye. In one embodiment, the method further comprises administrationby injection into the anterior chamber of the eye. In one embodiment,the method further comprises administration using an ocular insert. Inone embodiment, administration of the tolbutamide increases aqueoushumor production In one embodiment, administration of the tolbutamideincreases aqueous outflow In one embodiment, the glaucoma is normaltension open angle glaucoma or exfoliative glaucoma.

Various embodiments disclosed herein also include a method of diagnosinga disease in a subject, comprising: obtaining a biological sample fromthe subject; testing the biological sample for presence of a mutation inKCNJ11 gene or Kir6.2 protein and in the AQP9 gene or aquaporin-9protein; and diagnosing a disease in the subject if the biologicalsample tests positive for the presence of a mutation in Kir6.2 proteinor KCNJ11 gene and or the AQP9 gene. In one embodiment, the disease isglaucoma. In one embodiment, the mutation is a nonsense mutation, rs5215(Gtc/Atc), in the KCNJ11 gene, which replaces valine with isoleucine atposition 337 of the ATP-sensitive potassium channel subunit and a gainof function of the Kir6.2 protein. In one embodiment, the mutation is anonsense mutation, rs1867380 (Aca/Gca) in the AQP9 gene, which resultsin the substitution of threonine for alanine (279T/A) at position 279 ofthe aquaporin-9 protein and a gain of function. In one embodiment, themethod further comprises treating glaucoma by administering to thesubject a composition comprising a therapeutically effective amount oftolbutamide or a physiologically equivalent salt or solvate thereof anda pharmaceutically acceptable carrier.

Embodiments of the present disclosure also include a method ofmaintaining and/or improving eye health in a subject, comprising:administering to the patient a composition comprising a therapeuticallyeffective amount of tolbutamide or a physiologically equivalent salt orsolvate thereof, and a pharmaceutically acceptable carrier, whereintolbutamide is in the composition at a concentration of 0.1-0.9% (w/v).In one embodiment, the tolbutamide increases aqueous humor production byat least 150%. In one embodiment, the 0.4% tolbutamide increases aqueousoutflow by at least 350%. In one embodiment, the pharmaceuticallyacceptable carrier is an ophthalmically acceptable carrier.

Embodiments of the present disclosure also include a method forregulating aqueous humor outflow via the ciliary body/trabecularmeshwork/Schlemm's canal complex in an eye of a glaucoma patient, saidmethod comprising: administering to the eye composition comprising acompound that specifically inhibits the Kir6.2 KATP channel.

Various embodiments disclosed herein further include a method ofmaintaining and/or improving eye health in a subject, comprising:administering to the eye of the subject a composition comprising atherapeutically effective amount of a compound that reestablishes theopen/close probability requirements of the Kir6.2 KATP channel tonormalize aqueous production/outflow dynamics.

Embodiments of the instant disclosure also include a method of treatingocular hypertension in a normal or glaucomatous subject, comprising:administering to the patient a therapeutically effective amount of acomposition comprising tolbutamide, sulfonylurea, and/or glinide, or aphysiologically equivalent salt or solvate thereof, and apharmaceutically acceptable carrier. In one embodiment, the ocularhypertension is reduced by at least 20%. In one embodiment, thepharmaceutically acceptable carrier is an ophthalmically acceptablecarrier.

Other features and advantages of the invention will become apparent fromthe following detailed description, taken in conjunction with theaccompanying drawings, which illustrate, by way of example, variousembodiments of the invention.

DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in referenced figures. It isintended that the embodiments and figures disclosed herein are to beconsidered illustrative rather than restrictive.

FIG. 1 depicts, in accordance with embodiments herein, binding of³H-glibenclamide to bovine trabecular meshwork cells (2.5×10⁵ per 0.5 mlof reaction mixture) at various concentrations of ligand. Red:non-specific binding; green: specific binding. Shown is mean±SD.

FIG. 2 depicts, in accordance with embodiments herein, displacement of³H-glibenclamide bound to trabecular meshwork cells (green) and RIN-m5Fcells (red) by tolbutamide.

FIG. 3 depicts, in accordance with embodiments herein, effect ofglybenclamide on the efflux of ⁸⁶Rb from human trabecular meshwork cell.Glybenclamide inhibits ⁸⁶Rb efflux in a dose dependent manner reaching50% inhibition at 10 nM

FIG. 4 depicts, in accordance with embodiments herein, effect ofchlorpromazine on the efflux of ⁸⁶Rb from human trabecular meshworkcell. Chlorpromazine inhibits ⁸⁶Rb efflux in a dose dependent manner,but on a molar basis it is less effective than glybenclamide, reaching20% inhibition at 1 μM

FIG. 5 depicts, in accordance with embodiments herein, long-term effectof 0.4% tolbutamide treatment on IOP of a human subject. Patient 1suffered from high IOP with no visual field loss. On days 1 through 6IOP was measured at 9:00 A.M., 1 drop of 0.4% Tolbutamide suspended inbuffered PBS (pH 6.7) was instilled to the right eye and IOP measured at12:00 Noon and at 3:00 P.M. The patient was instructed to apply one dropof drug 10:00 P.M. to the right eye and come to the clinic each day tohave IOP measured. Note that during the first day the IOP remained highin this patient but decreased significantly during the next 5 days. Thebottles were color-coded and the patient was not aware of which bottlecontained the drug and which contained the vehicle. The patient and thetechnician measuring the patient's IOP was not aware of which bottlecontained the drug and which contained the vehicle

FIG. 6 depicts, in accordance with embodiments herein, Patient 2, whosuffered from glaucoma and had become refractory to Timolol, was treatedwith 0.4% Tolbutamide after a 3-week washout. On days 1 through 5, IOPwas measured at 8:00 A.M., 1 drop of 0.4% Tolbutamide suspended inbuffered PBS (pH 6.7) was instilled to the right eye and IOP measured atthe indicated times. A second drop was administered at 10:00 PM. Allmeasurements and drug administrations were done in the hospital. Thebottles were color-coded and the patient was not aware of which bottlecontained the drug and which contained the vehicle. The patient and thetechnician measuring the patient's IOP was not aware of which bottlecontained the drug and which contained the vehicle

FIG. 7 depicts, in accordance with embodiments herein, long-term effectof 0.4% tolbutamide treatment on IOP of glaucoma patients. Patients 3and 4 were given a bottle of drug and a bottle of control fluid(vehicle) and were instructed to instill one drop from one bottle in theright eye (drug) and one drop from the second bottle to the left eye(control) at 9:00 AM and at 10:00 PM. The patient was asked to come tothe clinic at 8:30 AM and at 5:00 PM each day to have the IOP measured.The bottles were color-coded and the patient was not aware of whichbottle contained the drug and which contained the vehicle. The patientand the technician measuring the patient's IOP was not aware of whichbottle contained the drug and which contained the vehicle

DETAILED DESCRIPTION

All references, publications, and patents cited herein are incorporatedby reference in their entirety as though they are fully set forth.Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Hornyak, et al., Introductionto Nanoscience and Nanotechnology, CRC Press (2008); Singleton et al.,Dictionary of Microbiology and Molecular Biology 3rd ed., J. Wiley &Sons (New York, N.Y. 2001); March, Advanced Organic Chemistry Reactions,Mechanisms and Structure 7th ed., J. Wiley & Sons (New York, N.Y. 2013);and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4th ed.,Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y. 2012),provide one skilled in the art with a general guide to many of the termsused in the present application. One skilled in the art will recognizemany methods and materials similar or equivalent to those describedherein, which could be used in the practice of the present invention.Indeed, the present invention is in no way limited to the methods andmaterials described.

Definitions

As used herein, the terms “ATP sensitive potassium channel,” “ATPsensitive K+ channel,” “KATP channel,” or “KATP channel” are usedinterchangeably and refer to a type of potassium channel that is gatedby ATP.

As used herein, the term “KATP channel activator” refers to a chemicalcompound that interacts with a KATP channel and (a) increases thebaseline activity of the KATP channel or (b) increases the activity thatthe KATP channel has while another compound is bound to the channel.

As used herein, the term “KATP channel inhibitor” refers to a chemicalcompound that interacts with a KATP channel and (a) decreases thebaseline activity of the KATP channel or (b) decreases the activity thatthe KATP channel has while another compound is bound to the channel.

As used herein, the term “subject” refers to a vertebrate, such as amammal, a fish, a bird, a reptile, or an amphibian. Thus, the subject ofthe herein disclosed methods can be a human, non-human primate, horse,pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The termdoes not denote a particular age or sex. Thus, adult and newbornsubjects, as well as fetuses, whether male or female, are intended to becovered. In one aspect, the subject is a mammal.

As used herein, the term “patient” refers to a subject afflicted with adisease, condition, or disorder. The term “patient” includes human andveterinary subjects. In some aspects of the disclosed methods, thesubject has been diagnosed with a need for treatment of an eye diseaseincluding, but not limited to glaucoma, dry eyes, and/or cataract.

As used herein, the term “pharmaceutically acceptable carrier,” refersto a pharmaceutically acceptable material, composition, or vehicle thatis involved in carrying or transporting a compound of interest from onetissue, organ, or portion of the body to another tissue, organ, orportion of the body. For example, the carrier may be a liquid or solidfiller, diluent, excipient, solvent, or encapsulating material, or acombination thereof. Each component of the carrier must be“pharmaceutically acceptable” in that it must be compatible with theother ingredients of the formulation. It must also be suitable for usein contact with any tissues or organs with which it may come in contact,meaning that it must not carry a risk of toxicity, irritation, allergicresponse, immunogenicity, or any other complication that excessivelyoutweighs its therapeutic benefits.

As used herein, the term “pharmaceutically acceptable excipient” refersto an excipient that is useful in preparing a pharmaceutical compositionthat is generally safe, non-toxic, and desirable, and includesexcipients that are acceptable for veterinary use as well as for humanpharmaceutical use. Such excipients may be solid, liquid, semisolid, or,in the case of an aerosol composition, gaseous.

Glaucoma

Glaucoma is currently treated with various drugs that are intended tolower elevated IOP, even though IOP affect only about 60% of glaucomapatients and only 20% of individuals with elevated IOP becomeglaucomatous. The inventors have now disclosed a method for treatingglaucoma that is not focused on lowering elevated IOP, but rather bymodifying the basic metabolic defect that causes glaucoma.

The metabolic defect that causes glaucoma is namely preventing the highextracellular potassium that results from a mutation in the KCNJ1 gene,(GTC/ATC, 337V/I) and the high extracellular lactate and other toxicsubstances, e.g. glutamate, that result from a mutation in the AQP9 gene(ACA/GCA, 279T/A). Treatment with tolbutamide and other sulfonylureasresults in very high outflow rates of aqueous humor essential to removeexcess lactate and other waste products, which in high concentrationsengenders a toxic acidic environment that over time results in retinalneuron degeneration.

In one embodiment, the inhibitor of the KATP channel is contemplated tobe tolbutamide. Sulfonylureas are a known treatment for diabetes.Diabetes patients are advised to take between 250 mg to 2 g ofsulfonylurea daily for the treatment of diabetes.

The inventors of this instant application have unexpectedly found thatvery low dosages of sulfonylureas, usually in microgram level oftolbutamide, treat glaucoma. For example, in one embodiment, theinventors treated glaucoma patients with a solution of 250 μgtolbutamide twice daily. This is a concentration that is 1000 timeslower that the minimal starting dose used to treat diabetes. At the lowconcentrations of sulfonylureas, particularly tolbutamide, contemplatedherein, the inventors found that glaucoma can be treated in the patientbut not diabetes. In other words, when a patient having both diabetesand glaucoma is administered tolbutamide at the concentrationscontemplated herein, glaucoma would be successfully treated, butdiabetes would not be successfully treated.

The inventors studied the scientific reasoning behind their unexpectedand surprising result. They found that sulfonylureas are used in thetreatment of diabetes because they allow the beta cells of the pancreasto release insulin. Specifically, when there is a high level of glucosein the blood, it is metabolized in the pancreas increasing the level ofATP, which causes the closure of the pancreatic ATP-sensitive potassiumchannel, depolarizes the beta-cell membrane, activates voltage dependentcalcium channels, and causes an influx of calcium that elicits insulingranule exocytosis. Since in diabetes the ATP sensitive potassiumchannel is not responsive to increased ATP, sulfonylureas are used toclose the ATP channel triggering the influx of calcium and insulingranule exocytosis as if there was a high level of ATP.

On the other hand, trabecular meshwork cells do not produce or storeinsulin. Therefore, it was surprising that that sulfonylureas, whichtrigger the release of insulin in diabetes, would also be useful as atreatment for glaucoma.

As disclosed herein, the inventors have developed compositions andmethods for regulating the production of aqueous humor. In oneembodiment, the production of aqueous humor is regulated byadministering an inhibitor of the Kir6.2 KATP channel. In someembodiments, the composition is formulated with a pharmaceuticallyacceptable vehicle or excipient selected from the group comprising ofophthalmically acceptable preservatives, surfactants, viscosityenhancers, penetration enhancers, gelling agents, hydrophobic bases,vehicles, buffers, sodium chloride, and water.

Various embodiments disclosed herein include an anti-aging compositionfor maintaining and/or improving eye health in a subject, comprising:administering to the eye of the subject a therapeutically effectiveamount of a compound that modulates the ATP-sensitive potassium (KATP)channel, specifically the channel isoform comprising four SUR2A/B orSUR1 and four Kir6.2 subunits, in which a specific mutation, rs5215, inglaucoma patients that decreases sensitivity to ATP, a gain of functionmutation in glaucoma patients, and a pharmaceutically acceptablecarrier. In one embodiment, the compound increases outflow of aqueoushumor. The inventors have shown that a mutation in the KCNJ11 gene(rs5215) of the KATP channel replaces valine for with isoleucine atposition 337 (V337I) of the Kir6.2 protein. The isoleucine for valinesubstitution has been shown to result in gain of function. Since KATPchannels are modulated by intracellular levels of ATP, i.e. ATP inhibits(closes) and ADP stimulates (open) KATP channels, the ATP:ADP ratio is amajor factor determining channel activity. As disclosed herein thepotential gain of function the inventors have shown in trabecularmeshwork implies that inhibition of the KATP channels required a higherthan normal concentration of ATP; however, with age metabolism slows andless ATP is produced resulting in the trabecular meshwork KATP channelsbeing in the open state for longer periods of time, reducing aqueousoutflow and increasing IOP, which is a major risk for glaucoma. Drugsthat inhibit the KATP channel restore the normal open/closed probabilitystate of the channel to establish the aqueous humor outflow that ispresent in normal, non-glaucomatous individuals. In one embodiment, thecompound is an inhibitor of the Kir6.2 KATP channel. In one embodiment,the compound is a glinide. In one embodiment, the compound is asulfonylurea. In one embodiment, the compound is selected from the groupconsisting of carbutamide, acetohexamide, chlorpropamide, tolbutamide,glipizide, gliclazide, glibenclamide, glyburide, glibornuride,gliquidone, glisoxepide, glyclopyramide, glimepiride, chlorpromazine,2,3-butanedione and hydroxydecanoic acid, or a physiologicallyacceptable salt or solvate thereof. In one embodiment, thepharmaceutically acceptable carrier is an ophthalmically acceptablecarrier. In one embodiment, the compound is administered in an amountbetween about 0.1 μg and about 10 mg. In one embodiment, the dosages areadministered from 1 to 4 times per day. In one embodiment, the compoundis administered by topical application to the eye. In one embodiment,the compound is administered by injection into the anterior chamber ofthe eye. In one embodiment, the compound is administered using an ocularinsert. In one embodiment, the subject is a mammal. In one embodiment,the subject is a human. In one embodiment, the subject is a glaucomapatient. In one embodiment, the glaucoma is normal tension open angleglaucoma. In one embodiment, the glaucoma is high tension open angleglaucoma. In one embodiment, the glaucoma is exfoliative angle glaucoma.In one embodiment, the subject is a cataract patient. In one embodiment,the composition is administered after cataract surgery.

In one embodiment, disclosed herein is a method of treating glaucoma ina patient, comprising: obtaining a biological sample from the patient;testing the biological sample for presence of a mutation in Kir6.2protein or KCNJ11 gene; and provided that the biological sample testspositive for the presence of a mutation in Kir6.2 protein or KCNJ11gene, administering to the patient a composition comprising atherapeutically effective amount of tolbutamide or a physiologicallyequivalent salt or solvate thereof and a pharmaceutically acceptablecarrier. In one embodiment, the tolbutamide is in a suspension orsolution at a concentration of 0.1-0.2%, 0.2-0.3%, 0.3-0.4%, 0.4-0.5%,0.5-0.6%, 0.6-0.7%, 0.7-0.8% (w/v). In one preferred embodiment, thetolbutamide is in a suspension or solution at a concentration of 0.4%(w/v). In one embodiment, the mutation is a nonsense mutation, rs5215,in the KCNJ11 gene. In one embodiment, the mutation is a V337I mutationin Kir6.2 protein. In one embodiment, the mutation is a nonsensemutation, rs1867380, in the AQP9 gene. In one embodiment, the mutationis a T279A mutation in aquaporin 9 protein. In one embodiment, thepatient further has type 2 diabetes. In one embodiment, thepharmaceutically acceptable carrier is an ophthalmically acceptablecarrier. In one embodiment, the dosages are administered from 1 to 4times per day. In one embodiment, the method further comprisesadministration by topical application to the eye. In one embodiment, themethod further comprises administration by injection into the anteriorchamber of the eye. In one embodiment, the method further comprisesadministration using an ocular insert. In one embodiment, administrationof the tolbutamide increases aqueous humor production by at least 100%,or more preferably at least 110%, or more preferably at least 120%, ormore preferably at least 130%, or more preferably at least 140%, or mostpreferably at least 150%. In one embodiment, administration of thetolbutamide increases aqueous outflow by at least 150%, or morepreferably at least 200%, or more preferably at least 250%, or morepreferably at least 275%, or more preferably at least 300%, or morepreferably at least 325%, or most preferably at least 350%. In oneembodiment, the glaucoma is normal tension open angle glaucoma orexfoliative angle glaucoma.

In various embodiments, the pharmaceutical compositions according to theinvention may be formulated for delivery via any route ofadministration. “Route of administration” may refer to anyadministration pathway known in the art, including but not limited toaerosol, nasal, oral, transmucosal, transdermal or parenteral.“Parenteral” refers to a route of administration that is generallyassociated with injection, including intraorbital, infusion,intraarterial, intracapsular, intracardiac, intradermal, intramuscular,intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal,intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous,transmucosal, or transtracheal. Via the parenteral route, thecompositions may be in the form of solutions or suspensions for infusionor for injection, or as lyophilized powders.

The pharmaceutical compositions according to the present disclosure canalso contain any pharmaceutically acceptable carrier.

The pharmaceutical compositions according to the present disclosure canalso be encapsulated, tableted or prepared in an emulsion or syrup fororal administration. Pharmaceutically acceptable solid or liquidcarriers may be added to enhance or stabilize the composition, or tofacilitate preparation of the composition. Liquid carriers includesyrup, peanut oil, olive oil, glycerin, saline, alcohols and water.Solid carriers include starch, lactose, calcium sulfate, dihydrate,terra alba, magnesium stearate or stearic acid, talc, pectin, acacia,agar or gelatin. The carrier may also include a sustained releasematerial such as glyceryl monostearate or glyceryl distearate, alone orwith a wax.

The pharmaceutical preparations are made following the conventionaltechniques of pharmacy involving milling, mixing, granulation, andcompressing, when necessary, for tablet forms; or milling, mixing andfilling for hard gelatin capsule forms. When a liquid carrier is used,the preparation will be in the form of a syrup, elixir, emulsion or anaqueous or non-aqueous suspension. Such a liquid formulation may beadministered directly p.o. or filled into a soft gelatin capsule.

The pharmaceutical compositions according to the present disclosure maybe delivered in a therapeutically effective amount. The precisetherapeutically effective amount is that amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentin a given subject. This amount will vary depending upon a variety offactors, including but not limited to the characteristics of thetherapeutic compound (including activity, pharmacokinetics,pharmacodynamics, and bioavailability), the physiological condition ofthe subject (including age, sex, disease type and stage, generalphysical condition, responsiveness to a given dosage, and type ofmedication), the nature of the pharmaceutically acceptable carrier orcarriers in the formulation, and the route of administration. Oneskilled in the clinical and pharmacological arts will be able todetermine a therapeutically effective amount through routineexperimentation, for instance, by monitoring a subject's response toadministration of a compound and adjusting the dosage accordingly. Foradditional guidance, see Remington: The Science and Practice of Pharmacy(Gennaro ed. 21st edition, Williams & Wilkins PA, USA) (2005).

Typical dosages of an effective composition can be in the rangesrecommended by the manufacturer where known therapeutic compounds areused, and also as indicated to the skilled artisan by the in vitroresponses or responses in animal models. Such dosages typically can bereduced by up to about one order of magnitude in concentration or amountwithout losing the relevant biological activity. Thus, the actual dosagewill depend upon the judgment of the physician, the condition of thepatient, and the effectiveness of the therapeutic method based, forexample, on the in vitro responsiveness of the relevant primary culturedcells or histocultured tissue sample, such as biopsied malignant tumors,or the responses observed in the appropriate animal models, aspreviously described.

In one embodiment, disclosed herein is a method of diagnosing a diseasein a subject, comprising: obtaining a biological sample from thesubject; testing the biological sample for presence of a mutation inKir6.2 protein or KCNJ11 gene; and diagnosing a disease in the subjectif the biological sample tests positive for the presence of a mutationin Kir6.2 protein or KCNJ11 gene. In one embodiment, the disease isglaucoma. In one embodiment, the mutation is a nonsense mutation,rs5215, in the KCNJ11 gene. In one embodiment, the mutation is a V337Imutation in Kir6.2 protein. In one embodiment, the mutation is anonsense mutation, rs1867380, in the AQP9 gene. In one embodiment, themutation is a T279A mutation in aquaporin 9 protein. In one embodiment,the method further comprises treating the disease by administering tothe subject a composition comprising a therapeutically effective amountof tolbutamide or a physiologically equivalent salt or solvate thereofand a pharmaceutically acceptable carrier.

In one embodiment, disclosed herein is a method of maintaining and/orimproving eye health in a subject, comprising: administering to the eyeof the subject a composition comprising a therapeutically effectiveamount of a compound that modulates the Kir6.2 KATP channel and apharmaceutically acceptable carrier. In one embodiment, the compoundincreases outflow of aqueous humor. In one embodiment, the compound isan inhibitor of the Kir6.2 KATP channel. In one embodiment, the compoundis a sulfonylurea. In one embodiment, the compound is selected from thegroup consisting of carbutamide, acetohexamide, chlorpropamide,tolbutamide, glipizide, gliclazide, glibenclamide, glyburide,glibomuride, gliquidone, glisoxepide, glyclopyramide, glimepiride,chlorpromazine, 2,3-butanedione and hydroxydecanoic acid, or aphysiologically acceptable salt or solvate thereof. In one embodiment,the pharmaceutically acceptable carrier is an ophthalmically acceptablecarrier. In one embodiment, the compound is administered in an amountbetween about 0.1 μg and about 10 mg. In one embodiment, the dosages areadministered from 1 to 4 times per day. In one embodiment, the compoundis administered by topical application to the eye. In one embodiment,the compound is administered by injection into the anterior chamber ofthe eye. In one embodiment, the compound is administered using an ocularinsert. In one embodiment, the subject is a glaucoma patient. In oneembodiment, the glaucoma is normal tension open angle glaucoma. In oneembodiment, the glaucoma is high tension open angle glaucoma. In oneembodiment, the glaucoma is exfoliative angle glaucoma. In oneembodiment, the subject is a cataract patient. In one embodiment, thecomposition is administered after cataract surgery.

In one embodiment, disclosed herein is a method for regulating aqueoushumor outflow via the ciliary body/trabecular meshwork/Schlemm's canalcomplex in an eye of a glaucoma patient, said method comprising:administering to the eye a compound that specifically modulates theKir6.2 KATP channel. In one embodiment, the regulation of aqueous humoroutflow is an increase in aqueous humor outflow. In one embodiment, thecompound is an inhibitor of the Kir6.2 KATP channel. In one embodiment,the compound is present in an ophthalmically acceptable carrier in anamount effective to increase aqueous outflow. In one embodiment, thecompound is administered in a dosage between about 0.1 μg and about 10mg of the compound. In one embodiment, the compound is administeredbetween 1 and 4 times per day. In one embodiment, the method furthercomprises administration by topical application to the eye. In oneembodiment, the method further comprises administration by injectioninto the anterior chamber of the eye. In one embodiment, the methodfurther comprises administration using an ocular insert. In oneembodiment, the Kir6.2 KATP channel inhibitor is used to increaseaqueous humor outflow resulting in lower intraocular pressure in hightension open angle glaucoma. In one embodiment, the Kir6.2 KATP channelinhibitor is used to increase aqueous humor outflow in normal tensionopen angle glaucoma. In one embodiment, the Kir6.2 KATP channelinhibitor is used to increase aqueous humor outflow in exfoliative angleglaucoma. In one embodiment, the Kir6.2 KATP channel inhibitor is usedto increase aqueous humor outflow resulting in lower IOP after cataractsurgery. In one embodiment, the compound is a sulfonylurea. In oneembodiment, the compound is selected from the group consisting ofcarbutamide, acetohexamide, chlorpropamide, tolbutamide, glipizide,gliclazide, glibenclamide, glyburide, glibomuride, gliquidone,glisoxepide, glyclopyramide, glimepiride, chlorpromazine,2,3-butanedione and hydroxydecanoic acid, and therapeutically equivalentsalts and derivatives thereof.

Embodiments of the present disclosure are further described in thefollowing examples. The examples are merely illustrative and do not inany way limit the scope of the invention as claimed.

EXAMPLES Example 1 Glaucoma

Glaucoma is the second leading cause of irreversible blindnessworldwide, and it is expected that 80 million people will suffer fromthis disease by the year 2020 [Quigley H A, Broman A T, 2006. Br JOphthalmol. 90: 262-267] and 111.8 million in 2040 (Tham Y-G et al.2014. Ophthalmology 121:2081-90). The glaucomas are classified asopen-angle (open iridocorneal angle), closed-angle (closed iridocornealangle), and developmental glaucomas. Glaucomas are divided into primaryand secondary types. Secondary glaucomas are diseases secondary toanother condition, such as exfoliation or pigment-dispersion syndrome,whereas glaucomas are diseases in which the retinal ganglion cellsdegenerate and aqueous outflow may diminish. Primary open-angle glaucomaincludes both adult-onset disease (occurring after 40 years of age) andjuvenile-onset disease (occurring between the ages of 3 and 40 years ofage). Primary open angle glaucoma (POAG) is the most common form ofglaucoma and is associated with the progressive loss of retinal ganglioncell axons, along with supporting glia and vasculature. The mechanismsof retinal ganglion cell degeneration, i.e. mitochondrial dysfunctionand oxidative stress, are similar to the mechanisms leading to theneural degeneration observed in Alzheimer disease [Chrysostomou V, etal. Oxidative stress and mitochondrial dysfunction in glaucoma. CurrOpin Pharmacol. 2013; 13:12-5; Tsolaki F, et al. Alzheimer's disease andprimary glaucoma; is there a connection? Clin Ophthalmol 2011;5:887-890; Lai S-W, et al. Glaucoma may be a non-memory manifestation ofAlzheimer's disease in older people. Intl Phychogeriatrics 2017;29:1535-1541]. Increased intraocular pressure (IOP) is present in 60-70%of patients with POAG, referred to as high tension glaucoma (HTG),whereas 30-40% of patients with POAG have IOP within normal limits,referred to as normal tension glaucoma (NTG).

The pathology shared by the heterogeneous group of glaucoma disorders ischaracterized by progressive optic nerve atrophy and retinal ganglioncell (RGC) death [Vohra R, Tsai J C, Kolko M, 2013. The role ofinflammation in the pathogenesis of glaucoma. Surv Ophthalmol58:311-320], which gradually lead to visual field loss. Although theresearch in the field of glaucoma is substantial, the pathologicalmechanisms involved in the onset and development of the disease arestill not completely understood. Neuronal degeneration in glaucoma mightbe due to a combination of molecular factors, such as compromisedretrograde axonal transport along the optic nerve, neurotrophindeprivation, increased oxidative stress, or excitotoxic stress caused bya glutamate impaired response [Madeira M E I, Boia R, et al. 2015.Contribution of microglia-mediated neuroinflammation to retinaldegenerative diseases. Mediators Inflamm. 2015:15; Almasieh M, Wilson AM, et al. 2012. The molecular basis of retinal ganglion cell death inglaucoma. Prog Retin Eye Res. 31:152-181].

Advanced age and elevated intraocular pressure (IOP) are the main riskfactors for the onset and progression of glaucoma. Nevertheless, 30-40%of patients with glaucoma present IOP values within the normal range[Sommer A et al. 1991. Relationship Between Intraocular Pressure andPrimary Open Angle Glaucoma Among White and Black Americans: TheBaltimore Eye Survey. Arch Ophthalmol 109:1090-1095; Fan N, Wang P, etal. 2015. Ocular blood flow and normal tension glaucoma. Biomed Res Int.2015:308505]; of particular note is the fact that glaucoma patients withnormal IOP appear to have more localized and central visual fielddefects for normal tension glaucoma (NTG) than high tension Glaucoma(HTG) [Thonginnetra O, Greenstein V C, Chu D, et al., 2010. Normalversus High Tension Glaucoma: A Comparison of Functional and StructuralDefects, J Glaucoma; 19: 151-157] suggesting that increased IOP is notessential for neuronal degeneration. Since elevated IOP is the onlymodifiable risk factor, therapeutic strategies target lowering of theIOP and include pharmacological treatments, surgical procedures, andlaser treatment. Although high intraocular IOP is considered as the mostimportant risk factor for the development of glaucoma, it is neithernecessary nor sufficient since in many patients RGC degenerationcontinues in spite of treatment to lower IOP [Edward Brubaker R F.Delayed functional loss in glaucoma. LII Jackson Memorial Lecture. 1996.Am J Ophthalmol. 121:473-483]; in fact, the risk of unilateral blindnessin patients with open-angle glaucoma treated to lower IOP is estimatedto be around 27% during a 20-year follow-up [Hattenhauer M G, Johnson DH, et al. 1998. The probability of blindness from open-angle glaucoma.Ophthalmology. 105:2099-2104.]

The fact that aqueous humor outflow is diminished significantly inglaucoma may engender a harmful environment in the eye and possibly inother neural structures, leading to altered metabolism and retinalganglion cell degeneration. Recent research points to structural,metabolic and functional glaucoma-driven changes in both the eye and thebrain [Murphy M C, Conner P I, Teng C Y, et al. 2016. Retinal Structuresand Visual Cortex Activity are Impaired Prior to Clinical Vision Loss inGlaucoma. Sci Rep. 6: 31464], and it appears that glaucoma deteriorationis already present in the eye and the brain before substantial visionloss can be detected clinically in patients [Wollstein G. et al. 2012.Retinal nerve fibre layer and visual function loss in glaucoma: thetipping point. Br J Ophthalmol 96, 47-52; Alasil T. et al. 2014.Correlation of retinal nerve fiber layer thickness and visual fields inglaucoma: a broken stick model. American journal of ophthalmology 157,953-959]. Some of the metabolic changes in glaucoma that may underlayits pathology are calcium disregulation [He Y, Ge, Tombran-Tink J, 2008.Mitochondrial Defects and Dysfunction in Calcium Regulation inGlaucomatous Trabecular Meshwork Cells. Investigative Ophthalmology &Visual Science. 49: 4912-4922], alterations in glutamate and glutamine[Hu R G, et al. 2012. Alterations of glutamate, glutamine, and relatedamino acids in the anterior eye secondary to ischemia and reperfusion.Curr Eye Res. 37:633-43] and alteration in lactate transport andmetabolism [Jovanović P, et al. Dehydrogenase and oxidative stressactivity in primary open-angle glaucoma aqueous humour. Bos J Basic MedSci 22010; 10: 84-88; Kolko M, et al. Lactate transport and receptoractions in retina: Potential roles in retinal function and disease.Neurochem Res 2016; 1229-1236]. Since aquaporin-9 plays a significantrole in transporting lactate into cells, a mutation in the AQP9 genethat would result in the loss of aquaporin-9 function would adverselyaffect retinal ganglion [Miki A, et al. Loss of aquaporin 9 expressionadversely affects the survival of retinal ganglion cells]. and otherretinal cells that are dependent on lactate as an energy source [HurleyJ B, Lindsay K J, Du J. Glucose, lactate, and shuttling of metabolitesin vertebrate retinas. J Neurosci Res. 2015; 93:1079-1092; Vohra R,Kolko M. Lactate: More Than Merely a Metabolic Waste Product in theInner Retina. Mol Neurobiol. 2020; 57:2021-2037]

Even though glaucoma is a defect in aqueous outflow, which may or maynot result in increased IOP, reduction of IOP for both high tension andnormal tension glaucoma is currently the only dependable pharmaceuticalapproach to the management of POAG. Therapeutic agents for POAGtreatment include prostaglandin analogs, β-adrenergic receptor blockers,αβ-adrenergic receptor blockers, α1-adrenergic receptor blockers,α2-adrenergic receptor agonist, and carbonic anhydrase inhibitors (Kwonet al., 2009, N Engl J Med 360:1113-1124; Abu-Amero, et al., 2015, IJMS16:28886-2891; Sommer A et al, 1991, Arch Ophthalmol 109:1090-1095)

Topical prostaglandin analogs (PAs) are the most frequently drugs usedto treat glaucoma. Used once a day PAs lower IOP by 25-30% and stabilizeit at a lower level by increasing uveoscleral outflow. PAs can havesignificant side-effects, such as conjunctival hyperemia, irreversibledarkening of the iris in people with multicolor irises, increasedperiorbital (eyelid) skin pigmentation, local irritation, itching, dryeye, blurred vision, periorbital fat atrophy, and in rare cases maycause uveitis or cystoid macular edema. Beta blockers lower IOP by20-25% with once- or twice-daily dosing by decreasing aqueous formation.Beta blockers are well tolerated topically and rarely cause localadverse effects, such as stinging, itching, redness and blurred vision.Beta blockers, however, even though administered locally to the eye canhave significant systemic side effects including dizziness, bradycardia,respiratory depression, masking of hypoglycemia, and interfering withthe treatment of asthma by beta2-agonists. The systemic side effectshave limited their use as first-line therapy. Carbonic anhydraseinhibitors (CAIs), like beta blockers, decrease IOP by decreasingproduction of aqueous humor. CAIS are very effective and decrease IOP by30-50%, but have many systemic adverse effects, which restrict theiruse. When any one drug does not lower the IOP to a safe level, acombination of 2 or more drugs are used to achieve the desired IOP;however, the side effects of drug combination are also additive. Whenpharmacological agents are no longer effective at lowering IOPsufficiently, surgical intervention is necessary in the form of lasertrabeculoplasty or implantation of devices to allow outflow of aqueoushumor.

It should also be noted that even when drugs are effective at loweringIOP, retinal ganglion cells (RGCs) continue to undergo apoptosis andconsequent vision loss progresses, albeit at lower rate. Current drugsfor glaucoma affect either aqueous humor production or thenonconventional uveoscleral outflow pathway. There are no drugsavailable that increase aqueous humor outflow via the conventionaltrabecular meshwork/Schlemm's canal pathway and there are no drugs thatincrease the formation of aqueous that is necessary to bring nutrientsto the eye to maintain a metabolically healthy environment. In fact,many of the drugs to treat glaucoma decrease aqueous formation, such asthe commonly used beta blockers and carbonic anhydrase inhibitors.Therefore, it would be desirable to provide drugs useful for the controlof intraocular pressure, particularly for the treatment of glaucoma andother disorders related to elevated intraocular pressure, where suchdrugs increase aqueous humor outflow via the conventional trabecularmeshwork/Schlemm's canal pathway and have fewer side effects whencompared to present drugs; such drugs would have an increased beneficialeffect if they would also bring nutrients into the eye by increasingaqueous humor formation. Drugs for the treatment of elevated IOP due toother conditions, such as surgical intervention for cataracts would alsobe desirable. Such drugs should be safe, non-toxic, and be amenable toincorporation in carriers and vehicles suitable for administration tothe eye, either topically, by injection, or by ocular insert. These andother objectives will be met by the methods and compositions of thepresent invention, as described in more detail hereinafter.

Example 2 KATP Channel

The ATP-sensitive K+(KATP) channel was first described by Noma (Noma A,1983, Nature, 305:147) in cardiac muscle and has since been identifiedin a number of cells and tissues (Meisheri K, et al, 1995, MolecularPharmacology 47:155; Schmid-Antomarchi H et al, 1987, BiophysicalResearch Communications 146:21; Spruce A, et al., 1987, Journal ofPhysiology 382:213; Niki I, Ashcroft S J, 1993, Neuropharmacology32:9510). KATP channels couple cell metabolism to electrical activityand thus regulate cell functionality, such as insulin secretion frompancreatic β-cells, transmitter release from brain neurons, and regulatethe cellular and extracellular water balance.

The KATP channels, members of the inward rectifying K+ channel family,are octameric complexes composed of four Kir6.x subunits and foursulfonylurea receptors (SUR) subunits (Shyng S, Nichols C, 1997, J GenPhysiol 110:655). The Kir6 subfamily is a member of the inward rectifierfamily and has two members, Kir6.1 and Kir6.2. SURs are members of theABC superfamily and comprise sulfonyl urea receptors SUR 1, SUR 2A andSUR 2B (Bryan J et al., 2007, Pflugers Arch—Eur J Physiol 453:703;Aittoniemi J et al., 2009, Philosophical Transactions of the RoyalSociety B: Biological Sciences 364:257). SURs, by themselves, perform norecognized function. Instead, they undergo association with heterologouspore-forming subunits to form ion channels, which they regulate. SURscontains two nucleotide-binding domains as well as low and high affinitybinding sites for sulfonylurea drugs and related compounds, such asglibenclamide and tolbutamide, which are potent inhibitors ofSUR-regulated channel activity. SURs are the target of sulfonylurea andglinide drugs used to treat diabetes mellitus type 2, neonatal diabetes,and some forms of congenital hyperinsulinemia. In the pancreaticβ-cells, binding of sulfonylureas and glinides to KATP channels induceschannel closure, causing membrane depolarization, which activatesvoltage-dependent Ca²⁺ channels in the β-cell plasma membrane and theresulting Ca2+influx triggers Ca²⁺-dependent insulin granule exocytosis(Ashcroft F, Rorsman P, 1989, Prog. Biophys. Molec. Biol, 54:87; ProksP, et al., 2002, Diabetes 51:S368).

KATP channel activity is thought to be regulated mainly by the metabolicactivity of the cell via changes in the concentrations of intracellularadenine nucleotides. Electrophysiological studies have suggested that,based on their kinetics and pharmacological properties, distinct typesof KATP channels can be detected in various tissues. These differenttypes of KATP channels appear to result from cell-specific expressionand the combination of different subunits. Indeed, two Kir6.x (Kir6.1,also known as KCNJ8, and Kir6.2, also known as KCNJ11) and two SURx(SUR1, also known as ABCC8, and SUR2, also known as ABCC9) subunits havebeen identified, and their various combinations can give rise tofunctional KATP channel subtypes (Seino S, Miki T, 2003 Progress inBiophysics and Molecular Biology 81:133). Six functional KATP channelshave been identified, specifically SUR1/KIR6.1, SUR1/KIR6.2,SUR2B/KIR6.1, SUR2A/Kir6.1, SUR2A/KIR6.2, and SUR2B/KIR6.2. Thesechannels have different sensitivities to ATP, channel openers andchannel inhibitors as well as tissue distribution.

The KATP channels are regulated by intracellular ATP such that it isspontaneously active in the absence of ATP and closed by increasing ATPconcentration in the cytoplasmic side of the membrane. The KATP channelsare not activated by intracellular Ca⁺², and gating of the channel isindependent of membrane potential. The channel is selective for K+, andit is selectively inhibited by sulfonylurea compounds and glinides. Allpharmacological sulfonylureas contain a central S-arylsulfonylureastructure with a p-substituent on the phenyl ring (R) and various groupsterminating the urea N′ end group (R2). As an example for chlorpropamidethe p-substituent (R) on the phenyl ring is chloride (Cl—) and thesubstituent at the N′ of urea (R2) is a propyl group. Pharmacologicalsulfonylureas include carbutamide, acetohexamide, chlorpropamide, andtolbutamide. gliclazide, glibenclamide, glyburide, glibornuride,gliquidone, glisoxepide, and glyclopyramide, glimepiride. A number ofother sulfonylureas are used as biopesticides because they can interferewith plant biosynthesis of the amino acids, valine, isoleucine, andleucine. Glinides are a heterogeneous class of insulin secretionstimulating agents that bind to the KATP channel and close the channel.Glinides bind to the sulfonylurea receptor with a lower affinity thansulfonylureas (Stephan D, Winkler M, Kühner P, Russ U, Quast U.Selectivity of repaglinide and glibenclamide for the pancreatic over thecardiovascular KATP Channels. Diabetologia 2006; 49:2039-2048)

Sulfonylureas and glinides, which target KATP channels, are a mainstayof diabetes therapy. KATP channels are hetero-octameric structurescomposed of four regulatory sulfonylurea receptor subunits (SURs) andfour Kir6.x subunits, the latter forming a central ion pore that permitsK+ efflux. The importance of SUR1 as a regulator of KATP channelactivity is exemplified by the fact that loss- and gain-of-functionmutations result in congenital hyperinsulinemia (HI) and neonataldiabetes, respectively. KATP channels play a key role in insulinsecretion both in response to glucose, the main physiological stimulus,and to sulfonylurea drugs that are used to treat type 2 diabetes.Loss-of-function mutations reduce KATP channel activity, producing apersistent membrane depolarization that leads to the activation ofvoltage-gated Ca²⁺ influx and continuous insulin secretion, irrespectiveof the blood glucose level. Conversely, gain-of-function mutationsprevent the channel from closing in response to metabolically generatedchanges in adenine nucleotides. Thus the β-cell remains hyperpolarizedeven when blood glucose levels rise, thereby keeping voltage-gated Ca²⁺channels closed and preventing Ca²⁺ influx and insulin secretion.Congenital HI is characterized by abnormal high levels of insulinsecretion despite severe hypoglycemia. A number of mutations in Kir6.2have been identified in familial early-onset type 2 diabetic probandsand their families. Of particular interest is that increased risk ofglaucoma is associated with diabetes duration, and fasting glucoselevels.

To investigate the effect of modulators of openers and blockers of theKATP channel in higher animals, the inventors tested their effect on theintraocular pressure in rabbits and discovered that 800 μg oftolbutamide, chlorpropanamide, glibenclamide and tolazamide decreasedIOP whereas diazoxide, KATP channel opener, increased IOP significantlywithin 1 hour. In cynomolgous monkeys, short-term, 1-hour studies inalso showed that 500 μg tolbutamide a KATP channel blocker, decreasedIOP Since blockers of the KATP channel are drugs that have been used forover 50 years to treat diabetes Type II, the inventors obtained ethicalboard permission to treat glaucoma patients with a solution of 250 μgtolbutamide twice daily, a concentration that is 1000 to 2000 timeslower that the minimal starting dose used to treat diabetes.Tolbutamide, a well-known and world-wide clinically approved drug totreat Type II diabetes, was used as the prototype blocker of the KATPchannel. It was unexpected that 1 drop of tolbutamide twice dailydecreased IOP for the 6 days of the study. Tolbutamide solutiondecreased IOP not only in glaucomatous patients, but also in patientswith elevated IOP after cataract surgery. More surprisingly andunexpected was the result that after 3 days, administration of one dropof 0.5% tolbutamide solution to the eye twice daily increased aqueousformation by approximately 150% and increased outflow via the trabecularmeshwork/Schlemm's canal by 350%; none of the drugs currently used totreat glaucoma increase aqueous formation as well as aqueous outflowwith a net outflow balance. These data, in conjunction with data showingthat tolbutamide administered topically to the eye twice daily decreasedIOP in 5 patients suffering with glaucoma and one patients with elevatedIOP after cataract surgery, clearly indicated that in humans, blockersand not openers of the KATP channel increase aqueous outflow and candecrease IOP.

Since type II diabetes is treated with sulfonylureas, the lowering ofIOP and modulation of aqueous humor dynamics by the sulfonylureatolbutamide could be inferred as a result of the treatment of theunderlying diabetes; however, such inference is erroneous consideringthat to lower IOP in rabbits and humans 2 drops/day of 0.5% tolbutamideare required, which is equal to a dose of 500 μg/day (1.0 ml equals 20drops) whereas treatment of diabetes with tolbutamide requires “250 mg-2g PO qDay or q8-12 hr; not to exceed 3 g/day; maintenance dose >2 g/dayseldom required”.(https://reference.medscape.com/drug/tolbutamide-342725) and “The usualstarting dose is 1 to 2 grams daily. This may be increased or decreased,depending on individual patient response”(https://www.drugs.com/pro/tolbutamide.html). It is obvious that thedose required to lower IOP and modulate aqueous dynamics (500 μg/day)will not treat diabetes (250 mg-2 grams/day).

Example 3 Glaucoma Treatment

Since current drugs to treat glaucomas and elevated IOP slow but do notprevent RGCs degeneration and do not increase sufficiently aqueous humoroutflow via the ciliary body/trabecular meshwork/Schlemm's canalcomplex, its natural outflow pathway, and in addition have significantside effects, there is an urgent need for novel useful drugs for thetreatment of glaucomas and elevated IOP by regulating aqueous humordynamics via the ciliary body/trabecular meshwork/Schlemm's canalpathway. Novel methods and compositions for treating glaucomas andintraocular pressure in the eye of a patient are presented in thisdisclosure. The compositions comprise compounds that bind to thesulfonylurea receptors moiety of the KATP channels closing the channels(channel blocker) and thus modulate cellular potassium efflux, increaseoutflow facility via the ciliary body/trabecular meshwork/Schlemm'scanal complex, and decrease IOP. The KATP-channel blockers compounds arepreferably sulfonylurea compounds, more preferably being selected fromthe group that include carbutamide, acetohexamide, chlorpropamide, andtolbutamide, gliclazide, glibenclamide, glyburide (also known asMicronase), glibornuride, gliquidone, glisoxepide, and glyclopyramide,glimepiride (also known as Amaryland or Glimiprime), and therapeuticallyequivalent salts and derivatives thereof, and are preferably present inthe compositions in concentrations from about 0.001% to 10% by weight.Non-sulfonyl urea compounds, however, have also been found to beeffective, such chlorpromazine, 2,3-butanedione, hydroxydecanoic acidand glinides.

Such compounds are delivered to the eye in an ophthalmically acceptablecarrier in an amount effective to increase aqueous humor outflow whetherexhibiting elevated IOP or normal IOP (normotensive glaucoma) and lowerIOP when administered to an eye having elevated intraocular pressure.Suitable administration methods include, but are not limited to topicalapplication, injection, and timed release using an ocular insert orequivalent formulation.

Example 4 Formulations

The methods and compositions of the present disclosure are intended fortreatment of impaired aqueous humor outflow. In some embodiments, theimpaired aqueous humor outflow is caused by glaucoma or other eyeconditions. In some instances the patient requiring treatment forimpaired aqueous humor outflow may also manifest IOP elevation in theeye. The patient may be human, or other mammals.

Glaucoma is a term which embraces a group of ocular diseasescharacterized by normal aqueous humor production by the ciliary body andimpaired aqueous humor outflow by the trabecular meshwork/Schlemm'scanal pathway. The impaired aqueous outflow may result in hypoxicstress, oxidative stress, elevated levels of excitatory amino acids,such as glutamate and aspartate, decreased neurotrophic factors, and inabout 60% of patients impaired aqueous outflow increases IOP. Theseconsequences of decreased outflow result in damage and eventual death ofretinal ganglion neurons, which is glaucoma. Glaucomas arewell-described in the medical literature. In addition to glaucoma, otherconditions in which disregulation of aqueous outflow results in elevatedintraocular pressure levels include cataract surgery, steroid treatment,and treatment with other drugs known to elevate intraocular pressure.The methods and compositions of the present invention are intended totreat all such conditions, and are not limited to glaucoma or dry eyesonly, in order to lower the intraocular pressure to avoid damage to theoptic nerve and retinal ganglion cells.

It is expected that other selective KATP channel inhibitors will beidentified in the future and that they will be useful in the methods ofthe present disclosure. KATP channels have been identified in many celltypes, e.g., cardiac cells, skeletal and smooth muscle, neurons andpancreatic β-cells. It is very likely that KATP channels are found inmany cells, and the data present in the Experimental section hereinafterindicate the presence of such an KATP channel in the trabecular meshworkcells of the eye. The inventors have shown that sulfonylurea compoundsbind to a receptor present in trabecular meshwork cells and the kineticsof binding are the same as the binding of sulfonylurea compounds topancreatic (3-cells. In addition, glybenclamide (a sulfonylurea) andchlorpromazine inhibit potassium efflux from trabecular meshwork cellsas indicated by ⁸⁶Rubidiun efflux.

The KATP channel inhibiting compounds will be administered to the eye inamounts and over a schedule effective to lower the intraocular pressureof the eye, when the intraocular pressure is elevated or when it isnecessary to lower the intraocular pressure to prevent damage to theoptic nerve or when it is necessary to increase the outflow facility ofthe aqueous humor. The amount of the compound required for such loweringwill depend on a number of factors, including degree of initial pressureelevation, condition of the patient, specific formulation, activity ofthe particular compound which is being administered, and the like, withexemplary amounts being in the range from about 50 μg to 5 mg per dose(i.e., single application of the composition), usually being from 250 μgto 1 mg per dose.

Topical compositions for delivering the KATP channel modulatingcompounds of the present invention will typically comprise the compoundpresent in a suitable ophthalmically acceptable carrier, including bothorganic and inorganic carriers. Exemplary ophthalmically acceptablecarriers include water, buffered aqueous solutions, isotonic mixtures ofwater and water-immiscible solvents, such as alkanols, aryl alkanols,vegetable oils, polyalkalene glycols, petroleum-based gels,ethyl-cellulose, carboxymethylcellulose, polyvinylpyrrolidones,isopropyl myristates, dextran, glycerin, dextran, hypromellose,polyethylene glycol, polysorbate, polyvinyl alcohol, povidone, orpropylene glycol, and the like. Suitable buffers include sodiumchloride, sodium borate, sodium acetate, gluconates, phosphates, and thelike.

The formulations of the present disclosure may also containophthalmically acceptable auxiliary components, such as emulsifiers,preservatives, wetting agents, thixotropic agents (e.g., polyethyleneglycols, antimicrobials, chelating agents, and the like). Particularlysuitable antimicrobial agents include quaternary ammonium compounds,benzalkonium chloride, phenylmercuric salts, thimerosal, methylparaben,propyl paraben, benzyl alcohol, phenylethanol, sorbitan, monolaurate,triethanolamine, oleate, polyoxyethlene sorbitan monopalmitylate,dioctyl sodiumsulfosuccinate, monothioglycerol, and the like.Ethylenediamine tetracetic acid (EDTA) is a suitable chelating agent.

The following formulations are exemplary of the compositions of thisdisclosure. These formulations are illustrative only and are notintended to limit the scope of this invention and should not be soconstrued.

FORMULA 1.

Component Amount Tolbutamide 10 μg to 20 mg Thimerosal 0.001% PhosphateBuffered Saline 1 mlFORMULA 2

Component Amount Tolbutamide 10 μg to 20 mg Hypromellose  0.4% SodiumChloride to 300 mOSm Hydrochloric acid/sodium hydroxide pH 6.7Thimerosal 0.001%FORMULA 3

Component Amount Glybenclamide 1 μg to 20 mg Sodium chloride 8 mg Boricacid 1 mg Benzalkonium chloride 0.1 mg   Hydrochloric acid/sodiumhydroxide pH 7.0 Water for injection (qs) 1 mlFORMULA 4

Component Amount Glybenclamide 1 μg to 20 mg Methyl paraben 1 mg Propylparaben 1 mg Sodium chloride 5 mg Water for injection (qs) 1 ml

Example 5 Experimental Results

Effect of Tolbutamide on IOP of Normal Cynomologus Monkeys:

Formulations of the KA channel inhibitor tolbutamidewere tested as asuspension and as a solution for its ability to lower intraocularpressure in normal cynomologus monkeys. For the suspension, 0.4%tolbutamide was prepared in NaCl/borate buffer (0.8 mg NaCl, 1.0 mgboric acid, pH 7.2, water to 1 ml; for the solution, 0.4% tolbutamidewas solubilized in 0.25 M NaOH and added to 0.4% hypromellose (HPMC) andthe pH adjusted to 6.7. Tonicity was adjusted to 300 mgOSm with NaCl andpreserved with 0.001% thimerosal. The suspension and solution weretested as follows: Monkeys were anesthetized with ketamine hydrochlorideand the baseline IOP determined. One drop of drug was administered tothe right eye and the IOP determined 1 hour later, while the animalswere still under anesthesia; the left eye served as a control. Twoanimals for each treatment were used. The results are presented inTable 1. The decrease in IOP in the untreated eye is the result ofanesthesia.

TABLE 1 IOP mmHg (% Change) pre-treatment 1 hour post-treatmentTreatment OD OS OD (treated) OS (untreated) Tolbutamide 0.4% 1 24 24 15(−37) 18 (−25) suspension 2 24 22 18 (−25) 19 (−14) Tolbutamide 0.4% 126 26 22 (−19) 23 (−12) solution 2 25 23 17 (−32) 17 (−26)

The Presence of KATP in Trabecular Meshwork Cells:

Aliquots of bovine trabecular meshwork were incubated on ice withvarious nM concentrations of ³H-glibenclamide. Non-specific binding wasdetermined as the residual binding in the presence of 20 μM non-labeledglibenclamide. After 2 hrs incubation, ³H-bound glibenclamide wasseparated from free ³H-glibenclamide on Whatman GF/F filters soaked inincubation buffer. Specific binding as calculated as the differencebetween binding in the absence and presence of non-labeled 20 nMglibenclamide (FIG. 1). The data presented in FIG. 1 shows thattrabecular meshwork cells have a receptor for glybenclamide and byanalogy these cells have a receptor for other sulfonylureas.

To define whether the sulfonylurea receptor in trabecular meshwork cellsis similar to the sulfonylurea receptor of pancreatic β-cells, theinventors compared the kinetics of displacement of ³H-glibenclamide bytolbutamide in bovine trabecular meshwork cells and RIN-m5F cells (aninsulinoma cell line derived from rat pancreatic islet cells (ATCC®CRL-11605™). The data in FIG. 2 shows that the displacement of³H-glibenclamide by tolbutamide from TM cells (green) and RIM-m5F (red)cells occurred with the same kinetics, suggesting that the receptors onthe two cell lines have similar pharmacological properties.

Effect of Tolbutamide on IOP in Human Subjects.

a. Effect of 1 Drop of 0.4% Tolbutamide.

Three patients with elevated IOP, due to exfoliative glaucoma, to POAGand to the increase in IOP that occurs in some patients following lensextraction, were treated with one drop of 0.4% tolbutamide solution. IOPwas measured at “0” time and at one hour intervals for 5 hours (whenpossible) by a nurse while the patient was in the hospital. The resultsshown in Table 2, expressed as mm of Hg, show that tolbutamide candecrease IOP significantly in all three conditions of elevated IOP.

TABLE 2 Effect of one drop of 0.4% Tolbutamide on IOP Human SubjectsPatient 1 2 3 Exfoliative Primary IOP Spike Glaucoma Open Angle afterLens Disease of OD Glaucoma Extraction OD OS OD OS OD “0” hours 48 20 3432 30 (Pre-treatment IOP) 1 hr 30 27 2 hrs 44 18 20 3 hrs 32 14 26 22 4hrs 30 14 22 20 5 hrs 30 14 22 20

b. Effect of 0.4% Tolbutamide on Human Subjects for an Extended Time.

To determine whether longer-term treatment with tolbutamide lowered IOPwithout any significant side effects, four patients with POAG patientwas treated with one drop of 0.4% tolbutamide twice daily (FIGS. 5-7) asshown in the figures. Vials containing drug and vehicle were color-codedby the manufacturing facility; the patient, the nurse and theinvestigator were not aware which vial contained the drug and which vialcontained the vehicle.

For patient 1, on days 1 through 6, IOP was measured at 9:00 A.M., 1drop of 0.4% Tolbutamide suspended in buffered PBS (pH 6.7) wasinstilled to the right eye and IOP measured at 12:00 Noon and at 3:00P.M. The patient was instructed to apply one drop of drug 10:00 P.M. tothe right eye and come to the clinic each day to have IOP measured. Notethat during the first day the IOP remained high in this patient, butdecreased significantly during the next 5 days.

As illustrated in FIG. 6 and Table 3, Patient 2, who suffered fromglaucoma and had become refractory to Timolol, was treated with 0.4%Tolbutamide after a 3-week washout. On days 1 through 5, IOP wasmeasured at 8:00 A.M., 1 drop of 0.4% Tolbutamide suspended in bufferedPBS (pH 6.7) was instilled to the right eye and IOP measured at theindicated times. A second drop was administered at 10:00 PM. Allmeasurements and drug administrations were done in the hospital, sincethe patient was admitted for an unrelated condition.

TABLE 3 IOP OD (treated) OS (Control) Day Time mm Hg % Change mm Hg %Change 1 4:00 PM 31 16 6:00 PM 21 −32 13 −19 6:30 PM 18 −42 18 +12 28:00 AM 16 −48 18 +12 4:00 PM 27 −13 18 +12 8:00 PM 28 −10 18 +12 3 8:00AM 20 −35 18 +12 4:00 PM 22 −29 16   00 8:00 PM 21 −32 14 −13 4 8:00 AM18 −42 19 +19 4:00 PM 22 −29 13 −19 8:00 PM 24 −23 15  −6 5 8:00 AM 17−45 — — 2:00 PM 23 −26 19 +19

Patients 3 and 4 were given a bottle of drug and one of vehicle and wereinstructed to instill one drop from one bottle in the right eye (drug)and one drop from the second bottle to the left eye (control) at 9:00 AMand at 10:00 PM. The bottles were color-coded and the patient was notaware of which bottle contained the drug and which contained thevehicle. The patient was asked to come to the clinic at 8:30 AM and at5:00 PM each day to have the IOP measured (FIG. 7).

Effect of 0.4% Tolbutamide on Aqueous Humor Outflow Facility:

The unexpected results in the 5 glaucoma patients definitely show thatinhibition of the ATP-sensitive potassium channel lowers IOP in openangle glaucoma patients, in exfoliative glaucoma patients as well as inpatients with high IOP due to surgical intervention. Since Chowdhury hasreported that activation of the ATP-sensitive channel promotes aqueousoutflow in rodents, the inventors determined whether the lowering of IOPin glaucoma patients would be via a different mechanism. Thus, theyinvestigated whether one drop of 0.45 tolbutamide would affect aqueousdynamics using fluorophotometry to measure aqueous production andoutflow.

Fluorophotometry.

To define the mechanism of action of tolbutamide, measurements ofaqueous dynamics were done on patient 2 (See FIG. 6). For this study IOPand rate of aqueous formation were measured at 9:00 A.M. The patient wasasked to apply one drop of drug to the right eye at 9:15 A.M. and one at10:00 P.M. for 3 days. On the morning of the fourth day IOP and rate ofaqueous formation were again measured.

TABLE 4 Effect of Tolbutamide on Inflow and Outflow of Aqueous Humor inan Ocular Hypertensive Patient. Outflow Facility Inflow (μl/min)(μl/min/mmHg) IOP (mmHg) OS OD OS OD OS OD Pre- 2.1 1.9 0.161 0.146 2222 Treatment Post- 1.2 3.0 0.137 0.50 18 15 treatment

Facility of Outflow (C) was calculated after Goldman:

${C = \frac{{Rate}\mspace{14mu}{of}\mspace{14mu}{formation}}{{I{OPt}} - {{Episcleral}\mspace{20mu}{IOP}}}},$where IOPt is the intraocular pressure in mmHg at the time the rate ofinflow is measured. Episcleral pressure for this patient was estimatedat 9 mgmHg.

The results (Table 4) showing that KATP channel inhibition increase bothproduction and outflow of aqueous were unexpected, considering thatother researchers (such as Chowdbury et al) have not only reported thatactivation of the KATP channel decreases IOP in animal models ofglaucoma and in perfused human anterior segment but that activation ofthe KATP channel by openers of the channels increase aqueous outflow.The present data shows that in glaucoma patients inhibition of the KATPchannel increase aqueous production by 100% and increases aqueousoutflow by 350%, suggesting that the KATP channel modulates themetabolic activity of the ciliary body/trabecular meshwork/Schlemm'canal complex. These data unquestionably show that KATP channelinhibition and not activation regulates aqueous humor dynamics.

Adverse Ocular Effects of Tolbutamide Administered as Drops to the Eye.

To determine whether tolbutamide has any adverse ocular side effects,patients were observed for symptom of ocular toxicity. Specifically,patients were monitored for discomfort, ocular pain, tearing,photophobia, erythema, swelling, discharge and scaling, palpebralconjunctival inflammation, bulbar conjunctival inflammation; limbalinflammation, corneal epithelial changes and focal stromal infiltrates.Symptoms were classified as 0=normal; 1=mild; 2=moderate; and 3=severe.All patients in the study were found to be free of severe symptoms atany time during the study. In all cases symptoms were classifies as“0-1”.

Example 6 Experimental Results

In one embodiment, the gene for the KATP channels is mutated.Specifically, the inventors isolate trabecular meshwork cells from donoreyes that had a history of glaucoma. The genes for aquaporin 9 (AQP9)and both genes for the components of the KATP channel, the pore formingsubunits, KCNJ8 and KCNJ11, and the sulfonylurea subunit, ABCC8 andABCC9, were sequenced and compared to the sequence to publicly availabledata to determine specific mutations. The methods of the presentdisclosure are intended for the diagnosis and identification ofindividuals at risk within a population.

The KATP channels, members of the inward rectifying K+ channel family,are octameric complexes composed of four Kir6.x subunits and four SURsubunits. The Kir6 subfamily is a member of the inward rectifier familyand has two members, Kir6.1 and Kir6.2. SURs are members of the ABCsuperfamily and comprise sulfonyl urea receptors SUR 1, SUR 2A and SUR2B. SURs, by themselves, perform no recognized function. Instead, theyundergo association with heterologous pore-forming subunits to form ionchannels, which they regulate. SUR1 contains two nucleotide-bindingdomains as well as low and high affinity binding sites for sulfonylureadrugs and related compounds. Drugs such as glibenclamide, tolbutamideand glinides are potent inhibitors of SUR-regulated channel activity.SURs are the target of sulfonylurea drugs and glinides used to treatdiabetes mellitus type 2, neonatal diabetes, and some forms ofcongenital hyperinsulinemia. In the pancreatic β-cells binding ofsulfonylureas and glinides to KATP channels induces channel closure,causing membrane depolarization, which activates voltage-dependent Ca²⁺channels in the β-cell plasma membrane and the resulting Ca²⁺ influxtriggers Ca²⁺-dependent insulin granule exocytosis.

The AQP9 gene encodes a member of a subset of aquaporins called theaquaglyceroporins. Aquaporin 9 protein, which belongs to the aquaporin'sfamily of water-selective membrane channels, allows passage of a broadrange of noncharged solutes including glycerol, lactate, urea, polyols,purines, and pyrimidines. This protein may also facilitate the uptake ofglycerol in hepatic tissue. The encoded protein may also play a role inspecialized leukocyte functions such as immunological response andbactericidal activity.

Alternate splicing results in multiple transcript variants.

Example 7 Mutation Results

In the eye intraocular pressure (IOP) is maintained by an equilibriumbetween aqueous production by the ciliary body and aqueous outflow viathe trabecular meshwork-Schlemm's canal complex. In approximately 60% ofprimary open angle glaucoma (POAG) patients IOP increases because of adecrease in aqueous outflow; however, in about 40% of glaucoma patientsthe IOP is normal. It is worth noting that in patients with high IOPless than 20% develop glaucoma 5 years after high IOP diagnosis (GordonM O, Toni V, Miglior S, et al. Ocular Hypertension Treatment StudyGroup; European Glaucoma Prevention Study Group, Validated predictionmodel for the development of primary open-angle glaucoma in individualswith ocular hypertension. Ophthalmology. 2007; 114:10-90). Therapeuticagents for the treatment of open angle glaucoma, whether patients havehigh or normal IOP include prostaglandin analogs, β-adrenergic receptorblockers, αβ-adrenergic receptor blockers, α1-adrenergic receptorblockers, α2-adrenergic receptor agonists, and carbonic anhydraseinhibitors. These drugs lower IOP be increasing uveoscleral outflow orby decreasing aqueous production. Rhepressa, a drug recently approveddecreases IOP at least in part by increasing outflow via the trabecularmeshwork (Kopczynski C C, Heah T. Netarsudil ophthalmic solution 0.02%for the treatment of patients with open-angle glaucoma or ocularhypertension. Drugs Today (Barc). 2018; 54:467-478.)

When pharmacological agents are no longer effective at lowering IOPsufficiently, surgical intervention is necessary. It should be notedthat even when drugs are effective at lowering IOP, retinal ganglioncells continue to undergo apoptosis and consequently vision lossprogression, albeit at lower rate.

Research of the inventors have shown that sulfonylureas and glinides,which are inhibitors of the ATP-sensitive potassium channels, lowerintraocular pressure significantly by increasing aqueous outflow via thenatural trabecular meshwork-Schlemm's canal pathway. As disclosedherein, their studies in humans have shown that 0.4% tolbutamide lowersintraocular pressure from 25-50% in primary open angle glaucomapatients, in exfoliative glaucoma patients and prevents the IOP spikethat occurs following cataract surgery. When outflow was examined viathe trabecular meshwork-Schlemm's canal complex the inventors found that0.4% tolbutamide increases aqueous production by 150% and increasesoutflow by 350%, a net 200% outflow increase. Since it is known thatwith normal aging aqueous humor production decreases, these resultssuggest that tolbutamide rejuvenates the ciliary body-trabecularmeshwork-Schlemm's canal complex. KATP channels are hetero-octamericcomplexes comprised of four pore-forming inward rectifier potassiumchannel subunits (Kir6.1 or Kir6.2) and four regulatory sulfonylureareceptor subunits (SUR1 or SUR2). Kir6.1 and Kir6.2 are encoded by thegenes KCNJ8 and KCNJ11, whereas SUR1 and SUR2 are encoded by ABCC8 andABCC9, respectively. The inventors sequenced the 4 genes in 9 glaucomapatients and found that in all 9 patients the KCNJ11 gene had a nonsensemutation (rs5215) that resulted in the substitution of valine forisoleucine at position 337 (337V/I) in the Kir6.2 protein resulting inloss of normal channel function; the loss of normal channel functionresulting from the V337V mutation indicates that in glaucoma patients,closure of the channel, which is normally mediated by ATP, requireshigher concentrations of ATP than the concentration required in normalpatients.

Research by the inventors has also shown the presence of aquaporin 9 inthe trabecular meshwork and ciliary body of humans; in addition, theinventors have discovered the AQP9 gene, which codes for aquaporin-9,has a potential loss-of-function mutation, rs1867380, which replaces theamino acid threonine with alanine (T279A) at position 279. Inindividuals that carry the mutation rs 5215 in the KCNJ11 gene, theslower metabolism and lower ATP concentration that occurs with age leadto the KATP-sensitive potassium channels remaining open and potassiumrelease into the extracellular space, which leads to increasedextracellular glutamate, lactate, and cell swelling as has been observedafter traumatic brain injury (Reinert M. et al High level ofextracellular potassium and its correlates after severe head injury:relationship to high intracranial pressure. J Neurosurg. 2000; 93:800-7)and restriction of aqueous outflow. The increased extracellularpotassium results in the release of lactate from trabecular meshworkcells increasing lactate in the aqueous from 410±89 μg/ml to over720±110 μg/ml (Jovanovic P, et al. Bos J Basic Med Sci 2010; 10:83-88).A second germline, non-synonymous mutation in the AQP9 gene (rs1867380,ACA→GCA) that replaces the amino acid threonine with alanine at position279 of the aquaporin results in a defective aquaporin-9, which does nottransport lactate to retinal ganglion cells, which is the major andpreferred source of energy and required for neuronal survival (Akashi A,et al. Aquaporin-9 expression is required for 1-lactate to maintainretinal neuronal survival. Neurosci Lett. 2015; 589:185-90). Sinceaquaporin-9 is essential for transporting lactate into neurons forenergy use, the loss of function will result in elevated levels ofextracellular lactate, which is also produced by astrocytes andtransported to retinal neurons by aquaporin-9 (Vohra R, Kolko M.Lactate: More Than Merely a Metabolic Waste Product in the Inner Retina.Mol Neurobiol. 2020, Epub ahead of print; Kolko M, et al. LactateTransport and Receptor Actions in Retina: Potential Roles in RetinalFunction and Disease. Neurochem Res. 2016; 41:1229-36). The accumulationof lactate, glutamate and waste products engenders a toxic environmentfor retinal neurons that over time leads to the neurodegenerationobserved in glaucoma.

The inventors discovered that in the trabecular meshwork and ciliarybody of human subjects there are two distinct KATP channels,specifically a channel that is composed of 4 Kir6.1 subunits and 4SUR-2A subunits and a KATP channel composed of 4 Kir6.2 and 4 SUR-2Asubunits, whereas the KATP in pancreas is compose of 4 subunits ofKir6.2 and 4 subunits of SUR1. The inventors have also localizedaquaporin-9 in the trabecular meshwork of human subjects (unpublished)and has been reported to be present in the retina (Hollborn M. et al.Expression of aquaporins in the retina of diabetic rats. Curr. Eye Res.2011; 36:850-856)

TABLE 4 Gene Mutations in Glaucoma Patients Protein Metabolic InhibitionGene Mutation Effect Result of K (ATP) KCNJ11* rs 5215 337 V/I ↑(K⁺)_(o)↓(K⁺)_(o) ↓(Lactate)_(o) (Gtc/Atc) (Gain of ↑(Lactate)_(o) ↑cellmetabolism function) ↑(glutamate)_(o) ↑ATP production AQP9** rs1867380279 T/A ↑(Lactate)_(o) ↑Aqueous production ((Aca/Gca) (loss of ↓(ATP in↑↑Aqueous outflow function) neurons) *KCNJ11 codes for the Kir6.2subunit of the K(ATP) channel **AQP9 codes for the aquaporin 9 channelthat transports lactate into neurons; lactate is an essential energysource and the loss of aquaporin9 adversely affects retinal ganglioncells (Miki A, et al, Am J Pathol 2013, 17271739; Akashi A, et alNeurosci Lett. 2015;589:185-90).

The data accumulated by the inventors indicate that glaucoma is aneurodegenerative disease that results from a toxic environment createdby excess lactate and glutamate as a result of mutations in two genes,specifically a mutation in the KCNJ11 gene and a mutation in the AQP-9gene. The KCNJ11 mutation results in high extracellular potassium ions,elevated lactate and glutamate, and elevated IOP resulting from cellswelling; the AQP-9 mutation results in an environment with high lactatesince the defective aquaporin-9 encoded by the mutated AQP-9 cannottransport lactate into neurons where it is serves as an energysubstrate, a source of NADH, and a scavenger of reactive oxygen species(ROS).

The various methods and techniques described above provide a number ofways to carry out the invention. Of course, it is to be understood thatnot necessarily all objectives or advantages described may be achievedin accordance with any particular embodiment described herein. Thus, forexample, those skilled in the art will recognize that the methods can beperformed in a manner that achieves or optimizes one advantage or groupof advantages as taught herein without necessarily achieving otherobjectives or advantages as may be taught or suggested herein. A varietyof advantageous and disadvantageous alternatives are mentioned herein.It is to be understood that some preferred embodiments specificallyinclude one, another, or several advantageous features, while othersspecifically exclude one, another, or several disadvantageous features,while still others specifically mitigate a present disadvantageousfeature by inclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability ofvarious features from different embodiments. Similarly, the variouselements, features and steps discussed above, as well as other knownequivalents for each such element, feature or step, can be mixed andmatched by one of ordinary skill in this art to perform methods inaccordance with principles described herein. Among the various elements,features, and steps, some will be specifically included and othersspecifically excluded in diverse embodiments.

Although the invention has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the embodiments of the invention extend beyond the specificallydisclosed embodiments to other alternative embodiments and/or uses andmodifications and equivalents thereof.

Many variations and alternative elements have been disclosed inembodiments of the present invention. Still further variations andalternate elements will be apparent to one of skill in the art. Amongthese variations, without limitation, are the selection of constituentmodules for the inventive compositions, and the diseases and otherclinical conditions that may be diagnosed, prognosed or treatedtherewith. Various embodiments of the invention can specifically includeor exclude any of these variations or elements.

In some embodiments, the numbers expressing quantities of ingredients,properties such as concentration, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

In some embodiments, the terms “a,” “an,” and “the” and similarreferences used in the context of describing a particular embodiment ofthe invention (especially in the context of certain of the followingclaims) can be construed to cover both the singular and the plural. Therecitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided with respectto certain embodiments herein is intended merely to better illuminatethe invention and does not pose a limitation on the scope of theinvention otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations on those preferred embodiments will become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Itis contemplated that skilled artisans can employ such variations asappropriate, and the invention can be practiced otherwise thanspecifically described herein. Accordingly, many embodiments of thisinvention include all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above citedreferences and printed publications are herein individually incorporatedby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that can be employed can be within thescope of the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention can be utilized inaccordance with the teachings herein. Accordingly, embodiments of thepresent invention are not limited to that precisely as shown anddescribed.

What is claimed is:
 1. A method of diagnosing and treating glaucomaassociated with V337I and T279A mutations in a patient in need thereof,comprising: determining a rs5215 GTC->ATC single nucleotide polymorphism(SNP) in the KCNJ11 gene, which changes the amino acid from valine toisoleucine at position 337 (V337I) of Kir6.2 subunit of theATP-sensitive potassium (KATP); determining a rs1867380 ACA->GCA SNP inthe aquaporin-9 (AQP9) gene, which changes the amino acid from threonineto alanine at position 279 (T279A) of aquaporin-9; diagnosing, upondetermination of the V337I and T279A mutations, glaucoma in the patienttreating glaucoma, upon diagnosing glaucoma, by administering to thepatient an inhibitor of the KATP channel.
 2. The method of claim 1,wherein the glaucoma is normal tension open angle glaucoma.
 3. Themethod of claim 1, wherein the glaucoma is high tension open angleglaucoma.
 4. The method of claim 1, wherein the glaucoma is exfoliativeglaucoma.
 5. The method of claim 1, wherein the inhibitor of the KATPchannel is formulated as an ophthalmically acceptable formulation. 6.The method of claim 1, wherein the inhibitor of the KATP channel is asulfonylurea.
 7. The method of claim 6, wherein the sulfonylurea istolbutamide.
 8. A method of claim 1, wherein the inhibitor of the KATPchannel is a glinide compound.
 9. A method of claim 1, wherein theinhibitor of the KATP channel is memantine.
 10. A method of claim 1,wherein the inhibitor of the KATP channel is chlorpromazine.
 11. Amethod of claim 1, wherein the inhibitor of the KATP channel isadministered topically to the eye.
 12. A method of claim 1, wherein theinhibitor of the KATP channel is administered as a slow-release ocularinsert.
 13. A method of claim 1, wherein the inhibitor of the KATPchannel is administered by injection into the eye.
 14. A method ofincreasing aqueous humor production in a patient in need thereof,comprising: identifying a patient having (a) nonsense mutation rs5215 inthe KCNJ11 gene, which changes the amino acid Valine to Isoleucine atposition 337 (V337I) of the Kir 6.2 protein and (b) nonsense mutationrs1867380 in the aquaporin-9 (AQP9) gene, which changes the amino acidthreonine to alanine at position 279 (T279A) of aquaporin-9; andadministering to the patient upon, identification of both of the V337Iand T279A mutations, a composition comprising tolbutamide or aphysiologically equivalent salt or solvate thereof and apharmaceutically acceptable carrier, wherein the administration oftolbutamide increases the aqueous humor production.
 15. A method ofincreasing intracellular and decreasing extracellular potassium in thetrabecular meshwork cells of glaucoma patients, comprising: identifyinga patient having (a) nonsense mutation rs5215 in the KCNJ11 gene, whichchanges the amino acid Valine to Isoleucine at position 337 (V337I) ofthe Kir 6.2 protein and (b) nonsense mutation rs1867380 in theaquaporin-9 (AQP9) gene, which replaces the amino acid threonine withalanine at position 279 (T279A) of aquaporin-9; and administering to thepatient upon, identification of both of the V337I and T279A mutations, acomposition comprising a sulfonylurea at a concentration of 0.1-0.9%(w/v) or a physiologically equivalent salt or solvate thereof and apharmaceutically acceptable carrier, wherein the administration of thesulfonylurea increases the aqueous humor production; whereinadministration of the sulfonylurea increasing intracellular anddecreasing extracellular potassium in the trabecular meshwork cells ofglaucoma patients.
 16. The method of claim 15, wherein the sulfonylureais tolbutamide.
 17. A method of claim 15, wherein the sulfonylurea isadministered topically to the eye.
 18. A method of claim 15, wherein thesulfonylurea is administered as a slow- release ocular insert.
 19. Amethod of claim 15, wherein the sulfonylurea is administered byinjection into the eye.
 20. A method of claim 16, wherein tolbutamide isadministered topically to the eye.
 21. A method of claim 16, whereintolbutamide is administered as a slow- release ocular insert.
 22. Amethod of claim 16, wherein tolbutamide is administered by injectioninto the eye.